title: Rationally designed coiled-coil DNA looping peptides control DNA topology.
abstract: Artificial DNA looping peptides were engineered to study the roles of protein and DNA flexibility in controlling the geometry and stability of protein-mediated DNA loops. These LZD (leucine zipper dual-binding) peptides were derived by fusing a second, C-terminal, DNA-binding region onto the GCN4 bZip peptide. Two variants with different coiled-coil lengths were designed to control the relative orientations of DNA bound at each end. Electrophoretic mobility shift assays verified formation of a sandwich complex containing two DNAs and one peptide. Ring closure experiments demonstrated that looping requires a DNA-binding site separation of 310 bp, much longer than the length needed for natural loops. Systematic variation of binding site separation over a series of 10 constructs that cyclize to form 862-bp minicircles yielded positive and negative topoisomers because of two possible writhed geometries. Periodic variation in topoisomer abundance could be modeled using canonical DNA persistence length and torsional modulus values. The results confirm that the LZD peptides are stiffer than natural DNA looping proteins, and they suggest that formation of short DNA loops requires protein flexibility, not unusual DNA bendability. Small, stable, tunable looping peptides may be useful as synthetic transcriptional regulators or components of protein-DNA nanostructures.
authors.affiliation: Department of Chemistry and Biochemistry, University of Maryland, College Park, MD 20742-2021, USA.
authors.forename: Daniel B
authors.initials: DB
authors.lastname: Gowetski
authors.forename: Erin J
authors.initials: EJ
authors.lastname: Kodis
authors.forename: Jason D
authors.initials: JD
authors.lastname: Kahn
mesh_terms: D001665:Binding Sites; D003500:Cyclization; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D016350:Leucine Zippers; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism
chemical_list: D010455:Peptides; D004247:DNA